Neuroblastoma is one of the most common solid tumors in children (Katzenstein, 1998). Available treatment is of limited utility for high-risk neuroblastoma and prognosis is therefore poor (Weinstein, 2003). Currently, children with high-risk neuroblastoma are treated with radiotherapy, dose-intensive cycles of multi-drug chemotherapy or, if patients responded poorly, with myeloablative dose of chemotherapy supported by stem cell rescue. Despite an aggressive treatment strategy, disease relapse occurs frequently and both short- and long-term toxicities, including treatment-related acute myeloid leukemia, occur in a significant percentage of disease survivors (Kushner, 1998) (Matthay, 1999). The high incidence of resistance of advanced stage neuroblastoma to conventional therapies has prompted investigators to search for novel therapeutic approaches.
Replication-competent viruses that replicate in tumor cells and lyticly kill them with limited side effects have been reported to have great potential in anti-tumor therapy (Ring, 2002; Thorne, 2005; Young, 2006; Parato, 2005). It has been suggested that antigen-presenting cells might internalize antigen released from virus infected tumor cells, leading to specific peptide presentation and generation of cytotoxic T lymphocyte (CTL), which, in turn, may facilitate tumor killing (Porosnicu, 2003; Berwin, 2001).
Poliovirus has recently been added to the list of viruses that hold promise as possible agents in tumor therapy (Gromeier, 2000; Ochiai, 2006). A non-enveloped, plus-stranded enterovirus of the Picornaviridae family, poliovirus replicates in the gastrointestinal tract causing little, if any, clinical symptoms. Rarely (at a rate of 10−2 to 10−3), the virus invades the central nervous system (CNS) where it targets predominantly motor neurons, thereby causing paralysis and even death (poliomyelitis). Poliovirus occurs in three serotypes all of which are defined in their amino acid sequences that specify the antigenic properties. That is, poliovirus type 1 has a capsid specifying serotype 1 antigenic sites.
Generally, poliovirus replicates efficiently in nearly all tumor cell lines tested, which has led to the suggestion that it may be suitable for the treatment of different cancers. However, the possibility that poliovirus can cause poliomyelitis calls for significant neuro-attenuation to avoid collateral neurological complications in cancer treatment. Additionally, there has been concern that the high coverage of anti-polio vaccination in early childhood in the U.S. and other countries may interfere with the application of poliovirus in tumor therapy.
Pathogenesis of poliovirus and of other neurotropic viruses can be controlled by translation (Gromeier, 1996; Gromeier, 2000; Mohr, 2005). In poliovirus, an exchange of the internal ribosome entry site (IRES) within the 5′-NTR with its counterpart from human rhinovirus type 2 (HRV2), another picornavirus, yielded viruses (called PV1(RIPO)) that are highly attenuated in CD155 tg mice (Gromeier, 1996; Gromeier, 1999) yet replicate efficiently and lytically in cell lines derived from solid glioma and breast cancer (Gromeier, 1996; Gromeier, 2000; Ochiai, 2004; Ochiai, 2006). However, PV1(RIPO) and PVS(RIPO), a derivative of PV1(RIPO), grow poorly in neuroblastoma cells (Gromeier, 1996; Gromeier, 2000).
With the exception of Raji cells, a Burkett's lymphoma cell line harboring a transcriptionally inactive CD155 gene (Solecki, 1997), wild type poliovirus kills all human tumor cells tested including neuroblastoma cell lines established from patients (Toyoda, 2004). Using the nude mice model, tumors of human origin can be successfully treated with neuroattenuated poliovirus strains, that is with PV(RIPO) derivatives (Gromeier, 2000), or with the Sabin vaccine strains (Toyoda, 2004). However, the lack of a possible immune response to the oncolytic agents mitigates the importance of the results. PVS(RIPO) is, in fact, under consideration for brain tumor therapy (Gromeier, 2000; Ochiai, 2006; Cello, 2008). However, as noted above, PVS(RIPO) replicates very poorly in human neuroblastoma cells, which disqualifies it from consideration in neuroblastoma therapy.
The whole genome synthesis of poliovirus (Cello, 2002) has produced, as by-product, the surprising observation that a point mutation (A103G) in a region between the cloverleaf and IRES, henceforth called “spacer region”, in the 5′-NTR (FIG. 1A) attenuated poliovirus neurovirulence 10,000 fold in CD155 tg mice (De Jesus, 2005). The A103G variant of poliovirus, named GG PV1(M), expresses a good replication phenotype in and kills human neuroblastoma cells (SK-N-MC) at 37° C. (De Jesus, 2005). This growth property of GG PV1(M) is different from that of PV(RIPO). However, GG PV1(M) is not useful in tumor therapy because the attenuating mutation A103G in the spacer region was unstable upon replication, and direct revertant variants rapidly emerge whose neurovirulence matches that of wt PV1(M) (De Jesus, 2005).
Novel therapeutic strategies are essential to improve the prognosis of patients with high-risk neuroblastoma. Neuroblastoma therapy with a poliovirus derivative may produce less toxicity often associated with chemotherapy and radiotherapy and complications such as second solid neoplasm, cardiopulmonary sequelae, renal dysfunction and endocrine consequences may not occur. The invention of a novel attenuated and stable poliovirus that will be effective in oncolytic treatment and cure of neuroblastoma is highly desirable.